Throughout this application various publications are referenced by partial citations within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
Since the purification of a pressor substance in blood serum termed serotonin (Rapport et al., 1947) and later identified as 5-hydroxytryptamine (5-HT) (Rapport, 1949), there has been a plethora of reports demonstrating that this indoleamine not only plays a role in the functioning of peripheral tissues but, indeed, performs a key role in the brain as a neurotransmitter. Certainly, the anatomical localization of serotonin and serotonergic neurons in both the peripheral and central nervous systems supports its role in such diverse physiologic and behavioral functions as pain perception, sleep, aggression, sexual activity, hormone secretion, thermoregulation, motor activity, cardiovascular function, food intake and renal regulation (For review see Green, 1985; Osborne and Hamon, 1988; Sanders-Bush, 1988; Peroutka, 1991). Taken together, it appears that serotonin plays an important role in homeostasis and in modulating responsiveness to environmental stimuli. Accordingly, studies demonstrating that abnormalities in the serotonergic system may be associated with disease states has created a drug development effort towards agents which may selectively modulate the function of serotonin (Glennon, 1990).
In relation to the characterization of physiologic or biochemical responses resulting from the release of serotonin are simultaneous investigation examining the receptor sites responsible for the actions elicited by the indoleamine transmitter. Following early in vitro pharmacological assays describing the existence of two different serotonin receptors, designated as D and M, in the guinea pig ileum (Gaddum and Picarelli, 1957), the advent of receptor binding technique in the 1970""s has brought to light during the last decade the diversity of 5-HT receptors existing in both the brain and peripheral tissues. Thus, although the concept of D and M receptors has not been invalidated, serotonin receptors not fitting either category have been identified using radioligand methods. To date using this technique, there appears to be four classes of serotonin receptors found in the brain: 5-HT1, 5-HT2, 5-HT3 and, putatively, 5-HT4 (Peroutka, 1991). Furthermore, 5-HT1 sites have been subclassified as: 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D (Hamon et al., 1990) and 5-HT1E (Leonhardt et al., 1989). Although a detailed characterization of the 5-HT1F binding site is lacking, extensive pharmacologic, biochemical and functional properties have clearly shown that the other four subtypes of 5-HT1 sites are receptors according to classical criteria.
During the last few years, the field of molecular biology has provided an important facet to receptor research by cloning these proteins and allowing more precise characterizations in isolated systems (Hartig et al,1990). This has been accomplished for the 5-HT1A (Fargin et al., 1988), 5-HT1C (Julius et al., 1988), 5-HT1D (Branchek et al., 1990) and 5-HT2 receptors (Pritchett et al., 1988). Thus, there is no doubt that these binding sites represent xe2x80x9ctruexe2x80x9d functional receptors. Indeed, the pharmacological characterization of serotonin receptors involved in various physiological or biochemical functions is a key component of drug development for the serotonergic system. As one can deduce from the diversity of serotonin binding sites, many targets are available for advancement in selective drug design. The coupling of molecular biological methods to pharmacological characterization particularly for cloned human receptors will open new avenues for pharmaceutical development which has not been previously explored.
This study is a pharmacological characterization of a serotonergic receptor clone with a binding profile different from that of any serotonergic receptor to date. In keeping with the nomenclature presently accepted for serotonin receptors, this novel site will be termed a 5-HT1F receptor based upon the fact that it possesses high affinity for the endogenous neurotransmitter, 5-HT.
This invention provides an isolated nucleic acid molecule encoding a human 5-HT1F receptor (Seq. I.D. No. 1).
This invention also provides an isolated protein which is a human 5-HT1F receptor (Seq. I.D. Nos. 2, 7).
This invention provides a vector comprising an isolated nucleic acid molecule encoding a human 5-HT1F receptor.
This invention also provides vectors such as plasmids comprising a DNA molecule encoding a human 5-HT1F receptor, adapted for expression in a bacterial cell, a yeast cell, or a mammalian cell which additionally comprise the regulatory elements necessary for expression of the DNA in the bacterial, yeast, or mammalian cells so located relative to the DNA encoding the 5-HT1F receptor as to permit expression thereof.
This invention provides a mammalian cell comprising a DNA molecule encoding a human 5-HT1F receptor.
This invention provides a method for determining whether a ligand not known to be capable of binding to a human 5-HT1F receptor can bind to a human 5-HT1F receptor which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a human 5-HT1F receptor with the ligand under conditions permitting binding of ligands known to bind to a 5-HT1F receptor, detecting the presence of any of the ligand bound to a human 5-HT1F receptor, and thereby determining whether the ligand binds to a human 5-HT1F receptor.
This invention also provides a method for determining whether a ligand not known to be capable of binding to the human 5-HT1F receptor can functionally activate its activity or prevent the action of a ligand which does so. This comprises contacting a mammalian cell comprising an isolated DNA molecule which encodes a human 5-HT1F receptor with the ligand under conditions permitting the activation of blockade of a functional response, detected by means of bioassay from the mammalian cell such as a second messenger response, and thereby determining whether the ligand activates or prevents the activation of the human 5-HT1F receptor functional output.
This invention further provides a method of screening drugs to identify drugs which specifically interact with, and bind to, the human 5-HT1F receptor on the surface of a cell which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a human 5-HT1F receptor with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, a human 5-HT1F receptor.
This invention also provides a method of screening drugs to identify drugs which interact with, and activate or block the activation of, the human 5-HT1F receptor on the surface of a cell which comprises contacting the mammalian cell comprising an isolated DNA molecule encoding and expressing a human 5-HT1F receptor with a plurality of drugs, determining those drugs which activate or block the activation of the receptor in the mammalian cell using a bioassay such as a second messenger assays, and thereby identifying drugs which specifically interact with, and activate or block the activation of, a human 5-HT1F receptor.
This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human 5-HT1F receptor.
This invention also provides a method of detecting expression of the 5-HT1F receptor on the surface of a cell by detecting the presence of mRNA coding for a 5-HT1F receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human 5-HT1F receptor under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the 5-HT1F receptor by the cell.
This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a human 5-HT1F receptor so as to prevent translation of the mRNA molecule.
This invention provides an antibody directed to a human 5-HT1F receptor.
This invention provides a transgenic nonhuman mammal expressing DNA encoding a human 5-HT1F receptor. This invention also provides a transgenic nonhuman mammal expressing DNA encoding a human 5-HT1F receptor so mutated as to be incapable of normal receptor activity, and not expressing native 5-HT1F receptor. This invention further provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a human 5-HT1F receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a 5-HT1F receptor and which hybridizes to mRNA encoding a 5-HT1F receptor thereby reducing its translation.
This invention provides a method of determining the physiological effects of expressing varying levels of human 5-HT1F receptors which comprises producing a transgenic nonhuman animal whose levels of human 5-HT1F receptor expression are varied by use of an inducible promoter which regulates human 5-HT1F receptor expression.
This invention also provides a method of determining the physiological effects of expressing varying levels of human 5-HT1F receptors which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of human 5-HT1F receptor.
This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a specific human 5-HT1F receptor allele which comprises: a. obtaining DNA of subjects suffering from the disorder; b. performing a restriction digest of the DNA with a panel of restriction enzymes; c. electrophoretically separating the resulting DNA fragments on a sizing gel; d. contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing to DNA encoding a human 5-HT1F receptor and labelled with a detectable marker; e. detecting labelled bands which have hybridized to the DNA encoding a human 5-HT1F receptor labelled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder; f. preparing DNA obtained for diagnosis by steps a-e; and g. comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step e and the DNA obtained for diagnosis from step f to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.
This invention provides a method of preparing the isolated 5-HT1F receptor which comprises inducing cells to express 5-HT1F receptor, recovering the receptor from the resulting cells and purifying the receptor so recovered.
This invention also provides a method of preparing the isolated 5-HT1F receptor which comprises inserting nucleic acid encoding 5-HT1F receptor in a suitable vector, inserting the resulting vector in a suitable host cell, recovering the receptor produced by the resulting cell, and purifying the receptor so recovered.
This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a receptor so as to prevent translation of the mRNA molecule.
This invention also provides a transgenic nonhuman mammal expressing DNA encoding a receptor.
This invention further provides a transgenic nonhuman mammal expressing DNA encoding a receptor so mutated as to be incapable of normal receptor activity, and not expressing native receptor.
This invention also provides a method of determining the physiological effects of expressing varying levels of a receptor which comprises producing a transgenic nonhuman animal whose levels of receptor expression are varied by use of an inducible promoter which regulates receptor expression.
This invention also provides a method of determining the physiological effects of expressing varying levels of a receptor which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of the receptor.
This invention further provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding the receptor and which hybridizes to mRNA encoding the receptor thereby preventing its translation.
This invention provides a method for determining whether a ligand not known to be capable of binding to a receptor can bind to a receptor which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding the receptor with the ligand under conditions permitting binding of ligands known to bind to a receptor, detecting the presence of any of the ligand bound to the receptor, and thereby determining whether the ligand binds to the receptor.